The in vivo micronucleus test, as performed in laboratory rodents, has gained widespread use as a short-term system to screen chemicals for clastogenic (chromosome-breaking) or aneugenic activity. The test is based on the observation that mitotic cells with either chromatid breaks or dysfunctional spindle apparatus exhibit disturbances in the anaphase distribution of their chromatin. After telophase, this displaced chromatin can be excluded from the nuclei of the daughter cell and is found in the cytoplasm as a micronucleus. Traditionally, micronuclei were scored in bone marrow preparations. An important advance came with the observation that micronucleated erythrocytes are not cleared from the blood of mice, thus allowing the analysis to be carried out more readily with peripheral blood samples. Erythrocytes are particularly well suited for evaluating micronuclei events since the nucleus of the erythroblast is expelled a few hours after the last mitosis yielding DNA deficient cells. Consequently, micronuclei are apparent in this cell population which is otherwise devoid of DNA. Treatment with clastogens and/or spindle poisons which cause genotoxic damage to stem cells results in the formation of easily detectable micronuclei in these young anucleated reticulocytes. These young anucleated cells are still rich in RNA and certain surface markers (e.g., CD71) and with appropriate staining can be distinguished from the mature normochromatic erythrocytes. From the bone marrow, these reticulocytes enter the bloodstream where they complete their evolution to RNA deficient normochromatic erythrocytes. By scoring micronuclei exclusively in the short-lived reticulocyte population, variation to micronuclei frequency can be attributed to a recent cell cycle, making the system amenable to acute exposure protocols.
Historically, micronucleus analyses have been conducted in rat bone marrow as opposed to rat peripheral blood because the rat spleen is able to capture and remove circulating micronucleated erythrocytes from peripheral circulation. However, recent reports have demonstrated the suitability of rat peripheral blood for micronucleus assessment when the analysis is restricted to the youngest of the immature RET population (i.e. Type I and II reticulocytes). The result is a sensitive index of genotoxicity (Wakata et al., 1998, Environmental and Molecular Mutagenesis, 21:136-143).
Analogous to microscopists restricting micronucleus scoring to Type I and Type II RETs based on RNA content, flow cytometric analysis can be limited to the youngest fraction of RETs based on the level of certain cell surface markers (e.g. CD71 or transferrin receptor expression). Fluorescent antibodies directed against the transferrin receptor are used to label the RET population. This immunofluorescent labeling procedure has been found to closely parallel the RET enumeration methods based on RNA content (Dertinger et al., 1996, Mutation Research, 371:283-292). Due to the direct relationship between RET maturity and CD-71 content, it is possible to characterize RETs according to age with this technique.
This modification to the mouse assay, which results in sensitive indications of genotoxicity in the rat peripheral blood compartment, has important implications for human biomonitoring. The spleen of the rat behaves very similarly to that of humans with regards to the effective scavenging of circulating micronucleated erythrocytes. Therefore, as with the rat, it is important to limit human peripheral blood micronucleus analyses to the youngest RETs (unless the individual is splenectomized).
An assay for micronucleated erythrocytes has applications as a system to evaluate nutrition or disease processes in humans. For example, in folate deficient humans, the frequency of micronucleated polychromatic erythrocytes appears to be associated with diet (Tucker et al., 1993, Mutat. Res. 301:19-26). Similarly, in splenectomized individuals, an increase in the frequency of micronucleated polychromatic erythrocytes has been associated with dietary factors, such as coffee and tea consumption (Smith et al., 1990, Cancer Res. 50:5049-5054; MacGregor et al., 1990, Proc. Natl. Acad. Sci.). Additionally, it is noted that various nucleoside analogues are being extensively used in the treatment of HIV-infected individuals. These analogues, used to inhibit replication of the virus, also significantly increase the frequency of micronucleated polychromatic erythrocytes in treated individuals (Phillips et al., 1991, Environ. Mol. Mutagen. 18:168-183. Thus, the frequency of micronuclei events in such individuals may be an additional indicator useful in monitoring nucleoside analog therapy.
The spontaneous background level of micronuclei in blood cells is usually quite low (approximately 2 micronuclei/1000 cells). The rarity of the micronuclei events coupled with the low throughput capacity of microscopic scoring procedures makes the conventional assay labor intensive and time consuming. The scoring operations are subject to human errors arising from the level of experience of each technician. Furthermore, assay sensitivity may be low due to the relatively small number of cells that are processed using the traditional microscopy-based scoring procedure. Manually scoring the slides for micronuclei takes weeks and is labor intensive. Practitioners of the art realize the need for automated methods to objectively and accurately score larger numbers of micronucleated cells thereby improving assay sensitivity and reliability.
At the present time in this art, the most rapid and accurate way to enumerate micronucleated erythrocytes in the total peripheral blood erythrocyte pool is by a flow cytometric method. One such method is disclosed in U.S. Pat. No. 5,229,265 (to the same Assignee hereof, the disclosure of which is herein incorporated by reference). In a flow cytometric method, cells pass in single file through a laser beam where a cell's fluorescence and light scatter properties are determined. In contrast to manual methods where only 1000-2000 cells per sample are scored, modern flow cytometers are capable of processing cells at rates in excess of 8,000 cells/second. By evaluating more cells, greater scoring accuracy is achieved. A considerable challenge has been to develop reliable automated methods for quantitating micronuclei events in peripheral blood and bone marrow reticulocytes. The advantage of restricting the analysis to these newly formed cells is that this population can highlight genotoxic or cytogenetic action resulting from acute exposures.
Classically, reticulocytes are divided into five populations which are defined by the staining pattern observed in the presence of RNA-precipitating dyes. Stains such as thiazole orange (Lee et al., 1986, Cytometry 7:508-516) and acridine orange (Seligman et al., 1983, Am. J. Hematology 14:57-66) are widely employed. However, in regards to a flow cytometry-based micronucleus assay, these and other RNA dyes are problematic. Since RNA dyes actually bind to DNA as well, overlapping signals tend to limit the resolution of micronucleated reticulocytes from micronucleated normochromatic erythrocytes. A flow cytometric method utilizing a dual dye combination consisting of thiazole orange and Hoechst 33342 has been described (Grawe et al., 1992, Cytometry 13:750-758). Thiazole orange stains the RNA component of the reticulocyte population, and Hoechst dye is used to label micronuclei. The dissimilar wavelengths necessary for the excitation of DNA and RNA dyes necessitates the use of a dual-laser flow cytometer.
Accordingly, there is a need in this art for a rapid, simple and accurate technique to determine the changes in the micronucleated cell populations in the blood and bone marrow cells caused by the action of genotoxic agents. Such a technique would desirably use reticulocyte and micronuclei-specific labels that are excited by a similar wavelength but exhibit significantly different emission spectra, thus enabling the use of a single-laser flow cytometer in a flow cytometric-based micronucleus assay.